Clostridium difficile is a common identified cause of antibiotic associated diarrhea, accounting for 15%-25% of cases (Bartlett, 1994, Clin. Infect. Dis., 18 (Suppl 4): S265-272). A more severe form of CDI has been identified. Many cases of this severe form of CDI have been shown to be caused by an “epidemic” strain of C. difficile, which has been characterized as “BI” by restriction enzyme analysis (REA), “NAP1” (North American Pulsed Field Type 1) by pulsed field gel electrophoresis, and “027” by PCR ribotyping. In addition, it has been characterized as “toxinotype III” (by REA of toxin genes). In prior years, the great majority of “non-epidemic” hospital strains of C. difficile belonged to toxinotype 0. This BI/NAP1/027 “epidemic” strain has been shown to be a hyperproducer of toxins A and B, to which is attributed the increased virulence of the epidemic strain. The tcdC gene, within the pathogenicity locus of the “epidemic” strain, appears to be a negative regulator of toxin A and B production.
Toxigenic strains of C. difficile commonly produce two large toxins, an enterotoxin; toxin A (TcdA) and a cytotoxin; toxin B (TcdB), to which disease symptoms are attributed. Without being bound by any particular theory, it is believed that these toxins are expressed efficiently during growth of C. difficile in response to an environmental stimulus. Their activities modulate numerous physiological events in the cell and are believed to contribute directly to disease. In humans the two toxins have been associated with pseudomembranous colitis and antibiotic associated diarrhea. Reported transmission occurs primarily in health care facilities, where exposure to antimicrobial drugs and environmental contamination by C. difficile spores are common.
Without being bound by any particular theory, it is believed that toxin A and toxin B are encoded by genes tcdA and tcdB. Both have been sequenced and are reportedly found in single open reading frames. Together with three additional genes (tcdC, tcdD, tcdE), they are reported to form a 19.6 kb chromosomal pathogenicity locus (Paloc) (8). Both open reading frames are large, with tcdA spanning a reported 8,133 nucleotides and tcdB being reported as 7,098 nucleotides in length. It is further believed that C. difficile toxigenic strains produced both toxin A and toxin B whereas nontoxigenic strains lacked both toxins (Rupnik et al. supra.; Lyerly et al., Clin. Micro. Rev., 1998, Jan. 1-18). Toxigenic reference strain VP1 10463 is an example of a strain producing TcdA and TcdB.
In determining the toxicity of a C. difficile strain, rapid determination is a central concern. If multiplex PCR can be applied, the process of determining the toxicity of a C. difficile strain will be radically improved. Multiplex PCR is a variant of PCR which enabling simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. Reported uses include genotyping applications where simultaneous analysis of multiple markers is required. It is also used in detection of pathogens. However, multiplex assays can be tedious and time-consuming to establish, requiring lengthy optimization procedures. Note is made of Clostridium difficile Toxins: Mechanism of Action and Role in Disease, Voth et al., Clinical Microbiology Reviews, (18)2: 247-263 (2005), the teachings of which are incorporated herein by reference.
Optimization of a determination of C. difficile type requires particular attention to many factors including primer design/selection, annealing temperature and extension time and temperature. Noted considerations in primer selection include selecting the length of individual primers, primer melting temperature and differential melting temperatures of all primers, purine:pyrimidine content; and degree of primer-primer interactions.
A related optimization issue is the adjusting of cycling conditions and buffer concentrations required for each primer pair. Annealing temperature is also a significant concern with unduly low annealing temperatures associated with the appearance of unspecific secondary products. Establishing an extension time is a significant consideration. The given of multiplex PCR is with more loci simultaneously amplified, then the pool of enzyme and nucleotides may become a key limiting factor. Thus, determination of time necessary for the polymerase molecules to complete synthesis is useful.
Reference is made to Rupnik, M. et al., J. Med. Microbiol, 2005, 54: 113-117; Voth, D. E. et al., Clinical Microbiol. Reviews, 2005, 18: 247-263; U.S. Pub. No. 20090208948 to Paquette et al., and U.S. Pub. No. 20090203021 to Cockerill et al., the teachings of which are incorporated herein by reference in their entirety as are all publications cited herein.